A flow-cytometer is a laser based bio-instrument that is employed in cell-counting, cell-sorting, and analysis of biological matter. In the flow-cytometer, a flow-cell carries cells via a sheath fluid. The sheath fluid aligns the cells into a single-file stream thereof. The single-file stream of cells is illuminated via a focused laser-beam. A beam-shaping optic is often employed to enlarge the focused beam in one axis to ensure illumination of the cell. Additionally, the beam-shaping optic can be used to homogenize the laser-beam in the same axis to ensure even illumination of the cells. As the cells pass through the focused laser-beam, laser light is scattered from the beam by the cells. The scattered light is collected by a detector system and analyzed. Results of the analysis are used to determine various properties and statistics, such as size, number, and the type of cells in the flow.
A flow-cytometer can employ fluorophores for facilitating the analysis. The fluorophores are fluorescent chemicals designed to attach to particular sites within the cells. The sites fluoresce when excited by a laser-beam of a particular wavelength, thereby acting like a tracer for the particular sites in which the fluorophores are attached. The fluorescence is collected by the detector system and provides additional data for analysis.
In order to excite multiple fluorophores in the flow-cytometer, multiple laser-beams are focused onto the single-file stream. At the focus, the laser-beams can be spatially separated or overlapped. The detector system of a flow-cytometer typically requires delivery of each focused, different-wavelength laser-beam to a specific location in the flow-cell.
One method of delivery of multiple different-wavelength beams to a flow-cell is by utilizing thin film filters such as dichroic filters. A common practice is to utilize forty-five degree long-pass filters in which shorter wavelengths are reflected from the filter, and longer wavelengths are transmitted through the filter. This technique requires at least one dichroic filter for two wavelengths, and an additional dichroic filter for every additional wavelength.
A problem with filter-based beam-delivery system is that multiple mirrors are often required in order to redirect the beams for focusing into the flow-cytometer. This results in complicated layouts and varied optical path lengths for any particular-wavelength beam. Additionally, each dichroic filter has multiple laser-beams transmitting or reflecting, which creates difficulties in aligning the filters individually and as a group.
As technology in flow-cytometry advances, new fluorophores are developed, which may require laser-beams of additional different wavelengths to be used in a flow-cytometer. A need exists for a beam-delivery system that can be easily customized for new flow-cytometer designs, and for upgrading existing flow-cytometers.